Abstract
The ability of ethanol and arachidonic acid (AA), as inducers of oxidative stress and key factors in alcoholic liver disease, to up-regulate alpha 2 collagen type I (COL1A2) gene expression was studied in a hepatic stellate cell line overexpressing the ethanol-inducible cytochrome P450 2E1 (CYP2E1) (E5 cells). A time- and dose-dependent induction in COL1A2 mRNA by ethanol or AA was observed that was prevented by diallylsulfide, a CYP2E1 inhibitor. Nuclear run-on experiments showed transcriptional activation of the COL1A2 gene by ethanol and AA. Catalase abrogated the increase in COL1A2 mRNA suggesting an H(2)O(2)-dependent mechanism. Cyclooxygenase-2 (COX-2) levels and production of prostaglandin E(2) upon addition of AA were elevated in the E5 cells. Incubation with NS-398, a COX-2 inhibitor, blocked the effect of AA, but not of ethanol, on COL1A2 expression suggesting that CYP2E1 activates COX-2 expression, and the oxidation of AA by COX-2 is responsible for the increase in COL1A2. Activity of a reporter construct driven by -378 base pairs of the proximal promoter region of the COL1A2 gene increased in E5 but not control cells and was further increased by ethanol or AA. These experiments link CYP2E1-dependent oxidative stress to induction of COX-2 and the actions of ethanol and AA on activation of collagen gene expression in hepatic stellate cells.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Allyl Compounds / pharmacology
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Animals
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Antioxidants / pharmacology
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Arachidonic Acid / pharmacology*
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Blotting, Northern
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Blotting, Western
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Cell Line
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Collagen / biosynthesis*
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Collagen Type I
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Cyclooxygenase 2
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Cyclooxygenase 2 Inhibitors
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Cyclooxygenase Inhibitors / pharmacology
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Cytochrome P-450 CYP2E1 / metabolism*
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Cytochrome P-450 CYP2E1 Inhibitors
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Dose-Response Relationship, Drug
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Electrophoresis, Polyacrylamide Gel
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Ethanol / pharmacology*
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Genes, Reporter
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Hydrogen Peroxide / metabolism
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Isoenzymes / metabolism
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Liver / drug effects*
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Models, Biological
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Nitrobenzenes / pharmacology
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Oxidative Stress
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Prostaglandin-Endoperoxide Synthases / metabolism
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Prostaglandins E / biosynthesis
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RNA, Messenger / drug effects
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Rats
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Recombinant Proteins / metabolism
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Sulfides / pharmacology
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Sulfonamides / pharmacology
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Time Factors
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Transcription, Genetic / drug effects
Substances
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Allyl Compounds
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Antioxidants
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Collagen Type I
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Cyclooxygenase 2 Inhibitors
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Cyclooxygenase Inhibitors
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Cytochrome P-450 CYP2E1 Inhibitors
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Isoenzymes
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Nitrobenzenes
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Prostaglandins E
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RNA, Messenger
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Recombinant Proteins
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Sulfides
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Sulfonamides
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N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide
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Arachidonic Acid
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Ethanol
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allyl sulfide
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Collagen
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Hydrogen Peroxide
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Cytochrome P-450 CYP2E1
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Cyclooxygenase 2
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Prostaglandin-Endoperoxide Synthases